Bottom Line:
It is currently unclear how A-lattice seams influence microtubule dynamic instability.Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly.On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

ABSTRACTNatural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

f3: Motility and shrinkage of A-lattice seam-enriched MTs.Segmentally labelled (Lemur-tailed) A-lattice-enriched MTs with alternating Alexa-680 (red) and Alexa-488 (green) labelling were created by co-assembly of Alexa-labelled pig brain tubulin and 50 μM Mal3FL with GMPCPP. MTs were imaged moving over a surface of kinesin-1 motor protein using fluorescence microscopy (a); scale bar, 5 μm. The internal MT stripes were used to determine the actual MT velocity and the velocity distribution of B- and A-lattice-enriched MTs were plotted (b). The mean velocities of B-lattice MTs were slightly slower at 675±9 nm s−1 (229) (mean±s.e.m. (n)) compared with A-lattice-enriched MTs at 707±6 nm s−1 (614) (Student t-test with Welch’s correction for unequal variances, P=0.0043). Gaussian fits to the distributions are shown in (b) with mean velocities of 660±12 nm s−1 (229) in A-lattice and 700±15 nm s−1 (614) in B-lattice (mean±s.d. (n)).

Mentions:
To control for the possibility that the kinesin surface interacts differently with B-lattice single seam and A-lattice-enriched multi-seam MTs, we checked for a difference in the kinesin-driven sliding velocity for the two types of MT. However, since in our assay the A-lattice-enriched GMPCPP MTs shrank rapidly while sliding over the kinesin surface, we could not immediately compare their velocity with that of the normal B-lattice single-seam MTs. We therefore used ‘lemur tail’ segmentally marked A-lattice-enriched GMPCPP MTs assembled using Mal3FL. Lemur tails are formed by the spontaneous end-to-end annealing of fluorescently labelled Alexa-488 pig brain tubulin MTs nucleated by Alexa-488 and Alexa-680 dual-labelled stabilized pig brain MT seeds (Fig. 3a). The mean sliding velocity of these lemur tail A-lattice-enriched GMPCPP MTs was similar to that of B-lattice single-seam GMPCPP MTs (707±6 nm s−1 (614) versus 675±9 nm s−1 (229) mean±s.e.m. (n); P=0.0043, t-test with Welch’s correction for unequal variances) (Fig. 3b), indicating that kinesins interact similarly with the two lattices.

f3: Motility and shrinkage of A-lattice seam-enriched MTs.Segmentally labelled (Lemur-tailed) A-lattice-enriched MTs with alternating Alexa-680 (red) and Alexa-488 (green) labelling were created by co-assembly of Alexa-labelled pig brain tubulin and 50 μM Mal3FL with GMPCPP. MTs were imaged moving over a surface of kinesin-1 motor protein using fluorescence microscopy (a); scale bar, 5 μm. The internal MT stripes were used to determine the actual MT velocity and the velocity distribution of B- and A-lattice-enriched MTs were plotted (b). The mean velocities of B-lattice MTs were slightly slower at 675±9 nm s−1 (229) (mean±s.e.m. (n)) compared with A-lattice-enriched MTs at 707±6 nm s−1 (614) (Student t-test with Welch’s correction for unequal variances, P=0.0043). Gaussian fits to the distributions are shown in (b) with mean velocities of 660±12 nm s−1 (229) in A-lattice and 700±15 nm s−1 (614) in B-lattice (mean±s.d. (n)).

Mentions:
To control for the possibility that the kinesin surface interacts differently with B-lattice single seam and A-lattice-enriched multi-seam MTs, we checked for a difference in the kinesin-driven sliding velocity for the two types of MT. However, since in our assay the A-lattice-enriched GMPCPP MTs shrank rapidly while sliding over the kinesin surface, we could not immediately compare their velocity with that of the normal B-lattice single-seam MTs. We therefore used ‘lemur tail’ segmentally marked A-lattice-enriched GMPCPP MTs assembled using Mal3FL. Lemur tails are formed by the spontaneous end-to-end annealing of fluorescently labelled Alexa-488 pig brain tubulin MTs nucleated by Alexa-488 and Alexa-680 dual-labelled stabilized pig brain MT seeds (Fig. 3a). The mean sliding velocity of these lemur tail A-lattice-enriched GMPCPP MTs was similar to that of B-lattice single-seam GMPCPP MTs (707±6 nm s−1 (614) versus 675±9 nm s−1 (229) mean±s.e.m. (n); P=0.0043, t-test with Welch’s correction for unequal variances) (Fig. 3b), indicating that kinesins interact similarly with the two lattices.

Bottom Line:
It is currently unclear how A-lattice seams influence microtubule dynamic instability.Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly.On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.

ABSTRACTNatural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe.